The neuron is remarkable in that upon the influx of Ca2+ it synchronizes vesicle fusion, and releases many quanta of neurotransmitters to the synaptic cleft in less than 1 msec. The fast synchronization is orchestrated by interactions between the core fusion machinery SNAREs and auxiliary proteins including a major Ca2+-sensor synaptotagmin 1 (Syt1) and a clamping factor complexin (Cpx). Because release underlies cognition and behavior, toxic agents that undermine the release of neurotransmitter might lead to the symptoms of neurodegenerative diseases such as Parkinson's and Alzheimer's. In this project we use innovative approaches to investigate the mechanism whereby the fusion machinery achieves the synchronization of vesicle fusion. To mimic the native membrane environment we prepare a nanodisc sandwich that harbors a single trans SNARE complex in the middle. Single molecule (sm)FRET and site-directed spin labeling (SDSL) EPR are used to characterize the interactions of SNAREs with auxiliary proteins in the chasm of two nanodiscs. In addition, the drastically improved single vesicle-vesicle fusion assay that can resolve docking, lipid mixing, fusion pore opening, and pore expansion steps, is used to delineate the intervention of regulatory factors onto individual fusion steps. Taken altogether, a comprehensive picture of how synchronization of vesicle fusion is choreographed by the interactions among individual components of the fusion machinery would emerge. For neurodegenerative diseases such as Parkinson's there is an emerging theme of pathophysiology that toxic misfolded oligomers are tampering with the vesicle fusion machinery, leading to disease symptoms. We use EPR to investigate the interaction between ?-synuclein and vesicle (v-)SNARE VAMP2 that takes place on the membrane surface. The outcomes of these investigations are expected to reveal new therapeutic targets for treating symptoms of the Parkinson's disease.